Research Article: Dissecting the Role of Critical Residues and Substrate Preference of a Fatty Acyl-CoA Synthetase (FadD13) of Mycobacterium tuberculosis

Date Published: December 21, 2009

Publisher: Public Library of Science

Author(s): Garima Khare, Vibha Gupta, Rakesh K. Gupta, Radhika Gupta, Rajiv Bhat, Anil K. Tyagi, Leonardo A. Sechi.

Abstract: Newly emerging multi-drug resistant strains of Mycobacterium tuberculosis (M.tb) severely limit the treatment options for tuberculosis (TB); hence, new antitubercular drugs are urgently needed. The mymA operon is essential for the virulence and intracellular survival of M.tb and thus represents an attractive target for the development of new antitubercular drugs. This study is focused on the structure-function relationship of Fatty Acyl-CoA Synthetase (FadD13, Rv3089) belonging to the mymA operon. Eight site-directed mutants of FadD13 were designed, constructed and analyzed for the structural-functional integrity of the enzyme. The study revealed that mutation of Lys487 resulted in ∼95% loss of the activity thus demonstrating its crucial requirement for the enzymatic activity. Comparison of the kinetic parameters showed the residues Lys172 and Ala302 to be involved in the binding of ATP and Ser404 in the binding of CoenzymeA. The influence of mutations of the residues Val209 and Trp377 emphasized their importance in maintaining the structural integrity of FadD13. Besides, we show a synergistic influence of fatty acid and ATP binding on the conformation and rigidity of FadD13. FadD13 represents the first Fatty Acyl-CoA Synthetase to display biphasic kinetics for fatty acids. FadD13 exhibits a distinct preference for C26/C24 fatty acids, which in the light of earlier reported observations further substantiates the role of the mymA operon in remodeling the cell envelope of intracellular M.tb under acidic conditions. A three-dimensional model of FadD13 was generated; the docking of ATP to the active site verified its interaction with Lys172, Ala302 and Lys487 and corresponded well with the results of the mutational studies. Our study provides a significant understanding of the FadD13 protein including the identification of residues important for its activity as well as in the maintenance of structural integrity. We believe that the findings of this study will provide valuable inputs in the development of inhibitors against the mymA operon, an important target for the development of antitubercular drugs.

Partial Text: Mycobacterium tuberculosis (M.tb), an intracellular pathogen, is exquisitely adapted for human parasitization [1]. It has evolved a number of distinct strategies to survive in the hostile environment of macrophages [2]. The drugs for the treatment of tuberculosis (TB) are available but the long and demanding regimens lead to erratic and incomplete treatment often resulting in the development of drug resistance. Hence, the importance of identification and characterization of new drug targets cannot be overemphasized.

mymA is an important operon of M.tb due to its involvement in the remodeling of the cellular envelope under stressful conditions and in the intracellular survival of the pathogen [8], [9], [10]. It is comprised of seven genes and the last gene encodes a Fatty Acyl-CoA Synthetase (Rv3089, fadD13). Due to essential role of mymA operon in the pathogenesis of M.tb, FadD13 represents an important drug target. A few other mycobacterial Acyl-CoA Synthetases namely MenE (involved in menaquinone biosynthesis) and FAAL28 and FAAL19 (involved in Fatty acid metabolism) have also been targeted for the development of antitubercular drugs and small molecule inhibitors against these enzymes have been developed [15], [16]. Besides, Fatty Acyl-CoA Synthetase inhibitor Triacsin C has been widely studied as a therapeutic agent for the treatment of artherosclerosis and certain kind of tumors [35], [36].



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